Chemical and enzymatic reactions are used to detect or quantitate the presence of certain substances in microbiological or other assays. Many of these tests rely on the development or change of color or fluorescence to indicate the presence or quantity of the substance of interest.
There are many examples of reactions used in Microbiology which rely on a color change. (Bascomb, Enzyme Tests in Bacterial Identification, 19 Meth. in Microbio. 105 (1987)). For example, a variety of organisms can be classified in large part by their pattern of fermentation, oxidation or assimilation of carbon sources. Fermentation of carbohydrates results in the production of acid which causes a decrease in pH. The drop in pH can be easily indicated by including a pH indicator like bromothymol blue or phenol red. With both indicators, acid conditions representing the fermentation of a particular carbohydrate result in a yellow color (changing from blue/green for bromothymol blue or pink/red for phenol red). The same approach can be adopted for a variety of carbohydrates, ranging from monosaccharides like glucose to polysaccnarides like inulin. In an analogous fashion, increasing pH can be followed. Assays for detecting the presence of decarboxylase and urease, and the ability to use malonate are based on an increase in pH, as indicated by a color change in the indicator.
Another approach to determine if an organism can degrade a particular substrate is to use a reagent which is capable of reacting with one or more of the intermediates or final products. For example, the detection of the reduction of nitrate to nitrite can be observed; if nitrite is formed, then a pink to deep red color will result when sulfanilic acid and alpha-napthylamine are added.
In contrast to the indirect detection of an enzymatic reaction illustrated by the nitrate/nitrite test, it is possible to use a synthetic analog of a natural substrate to directly indicate the presence of an enzyme. For example, methylene blue can be reduced under certain conditions by the action of reductase, resulting in a shift from blue to colorless.
Another test, the oxidase assay relies on the interaction of cytocnrome oxidase with N, N, N', N'-tetramethyl-p-phenylenedlamine producing a blue color.
Another example is the ability of microorganisms to degrade sulfur-containing amino acids as indicated by the production of H.sub.2 S. Typically, the organism is incubated with a high concentration of a sulfur-containing substrate (e.g. cysteine, cyscine) in an acid environment. The production of H.sub.2 S is indicated by the formation of a black precipitate in the presence of ferric ammonium citrate.
Although the use of colorimetric reactions is widespread there are limitations, especially in the sensitivity of detection. In order to improve sensitivity or, in the case of identification of microorganisms, to decrease the time required to obtain a result, fluorescence-based methods frequently are used. Unfortunately, either it is not possibe to develop a fluorescent equivalent to every assay or the reagents themselves are highly toxic and difficult to commercialize.
Additionally, the general principle of fluorescence quenching has been accepted as a way to follow enzymatic or chemical reactions. For example, Fleminger et al. synthesized intcamolecularly quenched fluorogenic substrates for the assay of bacterial aminopeptidase (P. Fleminger et al., Fluorogenic Substrates for Bacterial Aminopeptidase P and its Analogs Detected in Human Serum and Calf Lung, 125 Eur. J. Biochem. 609 (1982). In this case, the fluorescence of the aminobenzoyl group is quenched by the presence of a nitrophenylalanyl group. When the enzyme is present, the nitrophenylalanyl group is cleaved, with a concommitant increase in the sample's fluorescence. A variety of enzymes have been assayed by this type of procedure, including hydrolytic enzymes, other amino- and carboxypeptidases and an endopeptidase (Yaron et al., Intramolecularly Quenched Fluorogenic Substrates for Hydrolytic Enzymes, 95 Anal. Biochem. 228 (1979));(Carmel et al., Intramolecularly--Quenched Fluorescent Peptides as Fluorogenic Substrates of Leucine Aminopeptidase and Inhibitors of Clostridial Aminopeptidase, 73 Eur. J. Biochem. 617 (1977));(Carmel et al., An Intramolecularly Quenched Fluorescent Tripeptide as a Fluorogenic Substrate of Angiotensin-I-converting Enzyme and of Bacterial Dipeptidyl Carboxypeptidase, 87 Eur. J. Biochem. 265 (1978); (Florentin et al., A Highly Sensitive Fluorometric Assay for "Enkephalinase", a Neutral Metalloendopeptidase that Releases Tyrosine-Glycine-Glycine from Enkephalins, 141 Anal. Biochem 62 (1984).
In each instance, a synthetic substrate containing a quenching group and a fluorescing group was generated in order to detect the activity of the enzyme. An alternative to this approach would involve the synthesis of a resonance energy transfer pair of fluorescing groups on a substrate molecule. In this application, cleavage by the enzyme of one of the groups would result in a decrease in fluorescence, since the critical distance would be exceeded, eliminating the transfer of energy. However, these approaches are limited to specifically designed substrates.
Presently, the monitoring of color end-product in chemical and microbial reactions is usually achieved in either of two ways; 1) The detection of color end-product can be achieved by visual observation and estimated semi-quantitatively, or 2) the detection of color end-products or loss of color can be achieved by measuring the intensity of color instrumentally. Spectrophotometers that measure light absorbance are commonly used for this purpose.
When measuring the concentration of any substance it is advantageous to use one instrument or one principle of measurement, otherwise cost is increased.